Compositions and methods for the detection of zinc

ABSTRACT

The invention relates generally to compositions and methods for the detection of zinc. In particular, compositions and methods are provided to detect changes in cellular zinc concentration and to correlate them to cellular phenomena.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 14/503,914, filed Oct. 1, 2014, which a continuation of 14/078,743,filed Nov. 13, 2013, now abandoned, which claims priority to U.S.Provisional Patent Application 61/726,845, filed Nov. 15, 2012; and acontinuation-in-part of U.S. patent application Ser. No. 13/442,453,filed Apr. 9, 2012, now abandoned, each of which is incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM038784 and P01HD021921 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods for thedetection of zinc. In particular, compositions and methods are providedto detect physiological changes in zinc concentration (e.g.,extracellular, intracellular, etc.) and to correlate them to cellularphenomena.

BACKGROUND OF THE INVENTION

The biological functions of metal ions have traditionally been thoughtto be limited to structural and catalytic roles within proteins.However, metal ions are also known to play signaling roles at thecellular level. For example, the alkaline earth metal calcium is onemetal in which biological signaling roles are particularlywell-established. Periodic elevations in the intracellular concentrationof free calcium ions, also known as calcium transients or oscillations,are readily detected using fluorescent probes and are known to drive anumber of biological processes (Berridge et al. (2003) Nat Rev Mol CellBiol 4, 517-29.; herein incorporated by reference in its entirety). Thisis perhaps best illustrated in the egg, where repetitive calciumtransients are among the earliest observable events after fertilization(Lawrence et al. (1997) Development 124, 233-41.; herein incorporated byreference in its entirety). Several parameters of these calciumtransients, including total number, frequency, and amplitude influencewhich downstream developmental events are initiated (Ozil et al. (2006)Dev Biol 300, 534-44.; Ducibella et al. (2002) Dev Biol 250, 280-91.;Toth et al. (2006) Reproduction 131, 27-34.; herein incorporated byreference in their entireties). These cellular processes, which includecortical granule (CG) exocytosis and cell cycle progression can beinitiated in the absence of sperm by stimulatory agents that induceparthenogenesis (Kline & Kline. (1992) Dev Biol 149, 80-9.; Tahara etal. (1996) Am J Physiol 270, C1354-61.; Liu & Maller. (2005) Curr Biol15, 1458-68.; Madgwick et al. (2006) J Cell Biol 174, 791-801.;Battaglia & Gaddum-Rosse. (1987) Gamete Res 18, 141-52.; Ozil. (1990)Development 109, 117-27.; Zhang et al. (2005) Hum Reprod 20, 3053-61.;Tingen (2010) Science 330, 453; herein incorporated by reference intheir entireties). Interestingly, the normal pattern of calciumoscillations is disrupted in eggs matured under conditions which limitthe availability of the transition metal zinc (Kim et al. (2010) NatChem Biol 6, 674-81.; herein incorporated by reference in its entirety),suggesting that the physiologies of these metals are somehow connectedin the egg.

In a departure from its well-established role as an enzymatic cofactoror structure stabilizing agent, fluctuations in the total concentrationof intracellular zinc have recently been shown to contribute to theproper cell cycle regulation in maturing oocytes. Intracellular zinclevels increase by more than fifty percent and over 10¹⁰ ions per cellare accrued during the final stage of oocyte maturation, also known asmeiotic maturation (Kim et al. (2010) Zinc availability regulates exitfrom meiosis in maturing mammalian oocytes, Nat Chem Biol 6, 674-81.;herein incorporated by reference in its entirety). This significantcellular metal accumulation event occurs over a remarkably short timeinterval and is a physiological imperative, as insufficient accumulationof zinc leads to a premature meiotic arrest at telophase I instead ofmetaphase II (Kim et al. (2010) Nat Chem Biol 6, 674-81.). Thiszinc-dependent meiotic checkpoint arises, in part, becausezinc-insufficient eggs fail to reestablish maturation promoting factor(MPF) activity (Bernhardt et al. (2010) Biol Reprod, published ahead ofprint Nov. 10, 2010.; herein incorporated by reference in its entirety),which is necessary for eggs to set up and maintain meiotic arrest atmetaphase II.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a Zn-responsiveprobe comprising: (a) a Zn-binding group configured to coordinate one ormore Zn ions; (b) a signaling moiety configured to emit a detectablesignal; and (c) an attachment group, wherein said attachment group is achemically reactive with one or more functional groups. In someembodiments, the detectable signal of the signaling moiety is altered bycoordination of one or more Zn ions by the Zn-binding moiety. In someembodiments, the signaling moiety comprises a fluorophore. In someembodiments, the fluorescence intensity, emission spectra, and/orexcitation spectra of the signaling moiety is altered by coordination ofone or more Zn ions by the Zn-binding moiety. In some embodiments, theinteraction of the attachment group with a chemically reactivefunctional group results in covalent or stable non-covalent attachmentof the Zn-responsive probe to the functional group. In some embodiments,the Zn-binding group is attached to the signaling group, and thesignaling group is attached to the attachment group. In someembodiments, the one or more attachments are though a linker group. Insome embodiments, the functional group is attached to a surface.

In some embodiments, the present invention provides a method ofnon-invasively detecting Zn release and/or uptake by a cell or cellscomprising: (a) contacting a sample comprising a cell or cells with aZn-responsive probe (e.g., a Zn-responsive probe attached to a solidsurface); (b) detecting the signal emitted by the signaling moiety overtime; and (c) correlating signal emitted with Zn concentration (e.g.,intracellular or extracellular). In some embodiments, the samplecomprises one or more oocytes (e.g., human oocytes (e.g., for use in invitro fertilization), non-human oocyctes (e.g., oocytes of a non-humanprimate, domestic animal (e.g., feline, canine, etc.), agriculturalanimal (e.g., bovine, porcine, etc.)). In some embodiments, the presentinvention further comprises (d) removing the sample from theZn-responsive probe (e.g. separating the cell and the Zn-responsiveprobe). In some embodiments, removing the sample from the Zn-responsiveprobe (or the Zn-responsive probe from the sample) is facilitated by theattachment of the probe to a solid surface.

In some embodiments, the present invention provides a method ofdetecting the fertilization of an oocyte comprising: (a) contacting asample comprising an metaphase-II-arrested oocyte with a Zn-responsiveprobe (e.g., a Zn-responsive probe attached to a solid surface); (b)contacting the sample with sperm; (c) detecting a significant increasein extracellular Zn concentration based on a change in the signal of theZn-responsive probe; and (d) correlating the significant increase inextracellular Zn concentration to fertilization of the oocyte by thesperm. In some embodiments, methods further comprise (f) removing theoocyte from the Zn-responsive probe; and (g) implanting the fertilizedoocyte into a subject. In some embodiments, the Zn-responsive probe isattached to a surface. In some embodiments, removing the oocyte from theZn-responsive probe is facilitated by the attachment of the probe to asurface. In some embodiments, attachment of the Zn-responsive probe to asurface prevents uptake of the probe into the oocyte.

In some embodiments, the present invention provides a method ofevaluating the quality of an oocyte for fertilization comprising: (a)contacting a surface with a sample comprising the oocyte, wherein saidsurface displays a plurality of Zn-responsive probes (e.g.,Zn-responsive probes are attached to the surface); (b) detecting thesignal emitted by the Zn-responsive probes over time; (c) correlatingthe signal emitted with extracellular Zn concentration, wherein adecrease in extracellular Zn is indicative of an oocyte that is readyfor fertilization. In some embodiments, the oocyte is inprophase-I-arrest upon contacting with the surface. In some embodiments,methods further comprise (d) removing the oocyte from the Zn-responsiveprobe. In some embodiments, removing the oocyte from the Zn-responsiveprobe is facilitated by the attachment of the probe to a surface. Insome embodiments, attachment of the Zn-responsive probe to a surfaceprevents uptake of the probe into the oocyte.

In some embodiments, the present invention provides compositionscomprising a solid surface displaying one or more Zn-responsive probes,wherein said Zn-responsive probes comprise: (a) a Zn-binding groupconfigured to coordinate one or more Zn ions; and (b) a signaling moietyconfigured to emit a detectable signal. In some embodiments, thedetectable signal of the signaling moiety is altered by coordination ofone or more Zn ions by the Zn-binding moiety. In some embodiments, thesignaling moiety comprises a fluorophore. In some embodiments, thefluorescence intensity, emission spectrum, and/or excitation spectrum ofthe signaling moiety is altered by coordination of one or more Zn ionsby the Zn-binding moiety. In some embodiments, the composition furthercomprises a linker. In some embodiments, the linker connects theZn-binding group with the signaling moiety. In some embodiments, thelinker connects the Zn-binding group and/or the signaling moiety to thesolid surface. In some embodiments, the Zn-responsive probes areattached to the solid surface through the interaction of a attachmentgroup on the Zn-responsive probe and an anchor moiety on the solidsurface. In some embodiments, the solid surface is selected from thegroup comprising: a plate, bead, well, slide, biopolymer, cell surface,and tube.

In some embodiments, the present invention provides a method fordetection of Zn in a sample (e.g., by any suitable method). In someembodiments, a change in Zn concentration is detected. In someembodiment, Zn is detected by a Zn-responsive probe. In someembodiments, Zn is detected without a probe. In some embodiments,detection of Zn is correlated to a biological function or process (e.g.,oocyte fertilization). In some embodiments, a Zn-responsive probe isattached to a solid surface (e.g., bead, plate, slide, well, etc.). Insome embodiments, attachment of a Zn-responsive probe to a solid surfacefacilitates removal of the probe from a sample without contaminating thesample with Zn-responsive probe. In some embodiments, attachment of aZn-responsive probe to a solid surface facilitates removal of the samplefrom a surface (e.g., (tube, well, slide, etc.) without contaminatingthe sample with Zn-responsive probe. In some embodiments, attachment ofthe Zn-responsive probe to a surface prevents uptake of the probe intothe sample.

In certain embodiments, the present invention provides a Zn-responsiveprobe which has the following chemical structure:

wherein each of said Ras are individually selected from: H, CH₃, analkyl group, an O-alkyl group, an N-alkyl group, a linker group, anattachment group, and a linker group attached to an attachment group;wherein said R₁ is selected from: H, CH₃, an alkyl group, a linkergroup, an attachment group, and a linker group attached to an attachmentgroup; and wherein said R₂ is a Zn binding group.

In some embodiments, the Zn binding group has the following formula:

wherein A and B each seperately comprise at least one moiety selectedfrom an amine donor, an oxygen donor, and a sulfur donor.

In further embodiment, the Zn binding group has the following formula:

wherein A and B each seperately comprise at least one moiety selectedfrom a pyridine, an amine, an imidazole, a sulfonamide, a carboxylate, aphenol, an ether, an alcohol, a thiol, a thioether, and a thiophene.

In particular embodiments, the Zn binding group has the followingformula:

or a derivative of said formula that is able to chelate zinc.

In other embodiments, the Zn-responsive probe has the following chemicalstructure:

In some embodiments, each of the Ras are methyl groups. In otherembodiments, each of the R_(3s) are either hydrogen or an alkyl group.In further embodiments, R₁ is a methyl group or hydrogen. In furtherembodiments, R₁ and/or R₃ is an attachment group. In furtherembodiments, R₁ and/or R₃ is a linker attached to an attachment group.

In particular embodiments, the present invention provides methods ofnon-invasively detecting Zn release and/or uptake by a cell or cellscomprising: a) contacting a sample comprising a cell or cells with acomposition comprising a Zn-responsive probe, wherein the Zn-responsiveprobe is present in said composition at a concentration between 1×10⁻⁵ Mand 1×10⁻⁹ M; b) detecting the signal emitted by the signaling moietyover time; and c) correlating signal emitted with Zn concentration.

In certain embodiments, the sample comprises one or more oocytes. Inother embodiments, the Zn-responsive probe is attached to a surface. Inparticular embodiments, the concentration of the Zn-responsive probe isbetween 1×10⁻⁷ M and 9×10⁻⁸ M. In further embodiments, the cellcomprises a metaphase-II-arrested oocyte, wherein the method furthercomprises a step before step b) of contacting the sample with sperm, andwherein the detecting the signal comprises detecting a significantincrease in extracellular Zn concentration based on a change in thesignal of the Zn-responsive probe. In further embodiments, thecorrelating the signal comprises correlating the significant increase inextracellular Zn concentration to fertilization of the oocyte by thesperm. In other embodiments, the methods further comprise removing theoocyte from the Zn-responsive probe, and implanting the fertilizedoocyte into a subject. In particular embodiments, the Zn-responsiveprobe has one of the chemical structures shown above or in the figures.

In some embodiments, the present invention provides methods ofevaluating the quality of an oocyte for fertilization comprising: a)contacting a surface with a sample comprising the oocyte, wherein saidsurface displays a plurality of Zn-responsive probes; b) detecting thesignal emitted by the Zn-responsive probes over time; and c) correlatingthe signal emitted with extracellular Zn concentration, wherein adecrease in extracellular Zn is indicative of an oocyte that is readyfor fertilization.

In certain embodiments, the oocyte is in prophase-I-arrest uponcontacting with the surface. In other embodiments, the Zn-responsiveprobe is attached to a surface. In additional embodiments, theZn-responsive probe has one of the chemical structures shown above or inthe figures.

In particular embodiments, the present invention provides methods ofnon-invasively detecting Zn release and/or uptake by a cell or cellscomprising: a) contacting a sample comprising a cell or cells with acomposition comprising a Zn-responsive probe with the chemical structurerecited above or in the figures; b) detecting the signal emitted by thesignaling moiety over time; and c) correlating signal emitted with Znconcentration.

In certain embodiments, the Zn-responsive probes describes herein areused to detect or label zinc in cells as part of flow cytometry typeanalyses. In particular embodiments, the Zn-responsive probes are usedfor extracellular zinc tracking, such as for monitoring zinc exocytosis.In certain embodiments, the Zn-responsive probes are used atconcentrations to track zinc (e.g., inside biological samples) withminimal perturbation of normal physiology (e.g., at concentrations aslow as 1×10⁻⁷ M, 5×10⁻⁷ M, 1×10⁻⁸M and 5×10⁻⁸ M, or between 1×10⁻⁷ and9×10⁻⁸ M.

BRIEF DESCRIPTION OF THE DRAWINGS

The description provided herein is better understood when read inconjunction with the accompanying drawings, which are included by way ofexample and not by way of limitation.

FIG. 1, Panels a-e show images depicting how changes in extracellularzinc concentration are readily monitored with FluoZin-3 during in vitrofertilization. Rapid, repetitive increases in fluorescence intensitywere detected (a) by ROI analysis (denoted by white boxes in b-d). Thesespikes in fluorescent intensity involve the coordinated release of zincfrom the cell in a phenomenon defined as a “zinc spark.” Each zinc sparkcan be distinguished in the time-lapse series (b, 00:25:12) againstbackground fluorescence (representative example in c, 01:32:56).Successful fertilization was confirmed by the extrusion of a secondpolar body (d). Zinc sparks were also noted during strontiumchloride-induced parthenogenesis (e, 00:02:56).

FIGS. 2a-d show zinc sparks observed in eggs from two differentnon-human primate species, Macaca mulatta (a, b) and Macaca fascicularis(c, d). In both cases, a single calcium transient was induced byionomycin. Intracellular calcium was monitored with Calcium Green-1 AM,and extracellular zinc with FluoZin-3. The first panel begins at00:04:35 in c and 00:06:37 in d. Each subsequent panel represents animage acquired 4 s following the previous panel. Time is expressed ashh:mm:ss, wherein 00:00:00 represents the start of image acquisition.Scale bar=40 μm in c and d.

FIGS. 3a-c show that zinc sparks are polarized and are immediatelypreceded by intracellular calcium transients. Shortly followingactivation, zinc sparks occur around the egg cortex with the exceptionof a zinc spark-free region (a, 00:21:04, arrowhead). This spark-freeregion corresponds to the region containing the meiotic spindle, wherethe second polar body is extruded (a, 01:20:00, arrowhead). Eggactivation was confirmed by simultaneously monitoring intracellularcalcium oscillations with Calcium Green-1 AM every 4 s (b).Intracellular calcium increases immediately before a zinc spark, asevident when images were collected at a faster acquisition rate of every100 ms in an independent experiment (c). Time is expressed as hh:mm:ss,wherein 00:00:00 represents the start of image acquisition. In allcases, extracellular zinc was detected with FluoZin-3.

FIGS. 4a-e show that zinc sparks did not occur in the absence of calciumtransients. The membrane permeable derivative of FluoZin-3 (FluoZin-3AM) did not detect intracellular oscillations in zinc (a), suggestingthat those detected by Calcium Green-1 AM were specific to calcium.Insets are brightfield images taken at the beginning (left, first polarbody denoted as PB1) and end (right) of the acquisition period. In eachcase, the presence of the second polar body (PB2) at the conclusion ofimaging was observed, confirming successful egg activation anddevelopment. Zinc sparks were absent in the absence of an activatingagent such as strontium chloride (b), or in eggs treated with thecalcium-selective chelator BAPTA AM (c). Representative imagesillustrate that BAPTA AM-treated eggs neither extrude a second polarbody (b, 2 hrs postactivation, or hpf) nor form a pronucleus (c, 6 hpf).Intracellular calcium was detected by Calcium Green-1 AM (black line)and extracellular zinc was detected by FluoZin-3 (gray line) in b and c.Scale bar=75 μm in d and e.

FIGS. 5a-h show images depicting cortically polarized zinc ions in themouse egg. Total zinc, as detected by synchrotron-based x-rayfluorescence (a, b), is uniquely polarized in the unfertilized egg (a;i-iii represent replicates). This distribution is absent in the otheressential transition elements, such as iron (b; i-iii representreplicates). The range of each group of images is given units of μg/cm2.Labile (chelator-accessible) zinc, as detected by confocal microscopy(c-h), also has a hemispherical distribution in the live egg, asdetected by two chemically distinct zinc fluorophores: zinquin ethylester (c-e) and FluoZin-3 AM (f-h). Co-staining with a DNA marker (Syto64 in d, Hoechst 33342 in g) revealed that zinc was concentrated at thevegetal pole away from the meiotic spindle.

FIG. 6 shows total zinc content of the egg gradually decreases uponfertilization. Eggs and embryos were analyzed by XFM for theirtransition metal content. The mean zinc quota was highest in the in vivoovulated (IVO) MII egg and trended towards a decrease followingfertilization (see 2 and 6 hpf), reaching its lowest mean abundance inthe two-cell embryo. The iron and copper quotas were an order ofmagnitude less than zinc at all time points examined.

FIG. 7 shows intracellular zinc decreases in the activated egg.Corresponding to the onset of the zinc sparks (FluoZin-3, gray line),there is a gradual decrease in intracellular labile zinc that isdetectable with a membrane-permeable zinc fluorophore (FluoZin-3 AM,black line). Successful egg activation and development was confirmedusing simultaneous brightfield imaging, which revealed extrusion of asecond polar body (PB2). The first (0 min, left) and final (30 min,right) brightfield images are shown.

FIG. 8 shows optical sections from areas near the meiotic spindle (asdetected by DNA markers Syto 64 and Hoechst 33342), selected from thecomplete confocal Z-series and projected to show the corticallocalization of zinc-enriched vesicles as detected by two independentfluorophores, zinquin ethyl ester and FluoZin-3 AM. Merged images arealso shown for clarity.

FIGS. 9a-9b show exocytosis was inhibited by treating eggs withcytochalasin B prior to activation with strontium chloride. CytochalasinB did not affect the calcium oscillations (a) but blocked all but thefirst zinc spark in most cases (b).

FIG. 10 shows dose response of unfertilized eggs to increasingconcentrations of zinc pyrithione (ZnPT). Unfertilized eggs were treatedwith 10, 20, or 50 μM ZnPT for 15 min. A clear cytotoxic phenotype wasonly seen at 50 μM concentration. Thus, the effects of pyrithione aresignificantly below the threshold concentration (i.e., 500% lower) anddo not involve cytotoxicity, as pyrithione was used at a minimumconcentration (10 μM) and for a shorter duration (10 min).

FIGS. 11a-o show sustained elevation of intracellular zinc availabilityfollowing egg activation leads to reestablishment of metaphase arrest.Eggs were activated with strontium chloride (SrC12) then treated withzinc pyrithione (ZnPT) 1.5 hours later (a). At 6 h post-activation,control eggs form pronuclei (b, arrowheads) whereas the majority of eggstreated with ZnPT do not (c). When visualized by fluorescence, controleggs display decondensed DNA organized within a defined nucleus (d).F-actin is homogeneous around the egg's cortex (e) and α-tubulin remainsorganized as a spindle midbody remnant (f), which is visible between theegg and the second polar body (g, merged image). In contrast,ZnPT-treated eggs display condensed chromosomes (h) adjacent to an areaof concentrated, cortical F-actin (i). α-tubulin is organized in ametaphase-like configuration (j) around the chromosomes (k, mergedimage). This layout mirrors the subcellular arrangement in unfertilizedeggs (l-o), which also display condensed chromosomes (l) overlaid withan actin cap (m), surrounded by a metaphase spindle (n). Scale bar=80 μm(b, c) or 25 μm (d-o).

FIGS. 12a-e show perturbation of intracellular zinc availability causesactivation of the egg. Following 8 hours of culture, control eggsmaintain a metaphase II spindle (a, sp) with individual chromosomesaligned at the metaphase plate (b). In contrast, eggs exposed to 10 μMTPEN for the same period artificially activate as indicated by theformation of an intact pronucleus (c, pn) with decondensed chromatinsurrounding a nucleolus (d). The number of eggs displaying a pronucleusis significantly higher in the TPEN-treated group (e).

FIGS. 13a-d show the chemical structure of an exemplary Zn-responsiveprobe of the present invention (A, A zinc-chelating ligand containingfive potential zinc binding groups; B, BODIPY fluorophore; C, ethanolether based linker chain; D, amine group that enables attachment of thisfluorophore to carboxylate-modified surfaces via amide-couplingchemistries).

FIG. 14 shows a synthesis scheme for an exemplary Zn-responsive probe ofthe present invention.

FIG. 15A shows a formula for an exemplary Zn-responsive probe.

FIG. 15B shows the chemical structure of exemplary probe ZincBy-1.

FIG. 15C shows a schematic of the synthesis scheme for ZincBy-1described in Example 7 below.

FIG. 16 shows an absorbance spectra of 5 μM ZincBY-1 in 50 mM HEPES, 0.1M KNO₃, pH 7.2. “Apo” represents the absorbance spectrum in the absenceof zinc binding and “Zn” represents the absorbance spectrum in thepresence of 1 equiv of ZnSO₄.

FIG. 17 shows fluorescence spectra of 5 μM ZincBY-1 in 50 mM HEPES, 0.1M KNO₃, pH 7.2. “Apo” represents the fluorescence in the absence of zincbinding and “Zn” represents the fluorescence in the presence of 1 equivof ZnSO₄. 530 nm light was used for excitation and the maximumfluorescence occurs at 543 nm.

FIG. 18 shows the metal ion selectivity of ZincBY-1. Black barsrepresent ZincBY-1 fluorescence in the absence of zinc but in thepresence of Ca (5 mM), Mg (5 mM), Mn, Co, Fe, and Cu (all 10 μM). Greenbars represent the fluorescence in the presence of 1 equiv of zinc andCa (5 mM), Mg (5 mM), Mn, Co, Fe, and Cu (all 10 μM).

FIG. 19 shows a job plot of ZincBY-1, shown as a plot of mole fractionof ZincBY-1 vs. fluorescence intensity. The sum of the concentrations ofZincBY-1 and ZnSO₄ was held constant at 10 μM. The maximum at 0.5molefraction supports a 1:1 ZincBY-1/Zn binding ratio.

FIG. 20 shows a determination of ZincBY-1 dissociation constant (KD).ZincBY-1 fluorescence was measured in the presence of EGTA-bufferedsolution of Zn²⁺. Data was fit to a one-site binding model and yielded aK_(D)=4.4×10⁻⁹ M with a Hill coefficient of ˜2.

FIG. 21 shows the pH sensitivity of ZincBY-1. Normalized fluorescenceintensity of ZincBY-1 in the absence (Apo) and presence (Zn) of zinc atvarious pH values. Little change in fluorescence is observed for the Apoprobe over the range of pH values. Fluorescence from the Zn-bound probeis largely unchanged within the physiologically relevant pH range (pH4-7).

FIG. 22 shows ZincBY-1 is capable of labeling zinc-rich punctae in themammalian egg at 50 nM concentration. Image is a 3D reconstruction of az-stack of an MII mouse egg that has incubated in 50 nM ZincBY-1 tolabel zinc (green) and 0.1 mg/mL Hoechst to label DNA (blue). Scalebar=20 μm.

FIG. 23A shows exemplary formula for a Zn binding group.

FIG. 23B shows exemplary amino donor moieties that could be used for, oras part of, A and/or B in the formula shown in FIG. 23A.

FIG. 23C shows exemplary oxygen donor moieties that could be used for,or as part of, A and/or B in the formula shown in FIG. 23A.

FIG. 23D shows exemplary sulfur donor moieties that could be used for,or as part of, A and/or B in the formula shown in FIG. 23A.

DETAILED DESCRIPTION

The invention relates generally to compositions and methods for thedetection of zinc (Zn). In particular, compositions and methods areprovided to detect changes in extracellular and/or intracellular zincconcentration and correlate them to cellular phenomena. Experimentsconducted during development of embodiments of the present inventiondemonstrated the in vivo detection of changes in cellular (e.g.,extracellular, intracellular) zinc levels and correlation of thoselevels to cellular events. In some embodiments, detection of changes incellular (e.g., extracellular, intracellular) zinc levels provides amarker for the occurrence of cellular events (e.g., fertilization). Insome embodiments, the present invention provides reagents (e.g., probes)for the in vivo and/or in vitro measurement of Zn levels or changesthereof. In some embodiments, the present invention provides methods fordetection, measurement, and/or identification of changes in cellular Znlevels. In some embodiments, the present invention provides compositionsand methods for detecting changes in Zn levels (e.g., intracellular,extracellular, non-cellular) and correlating such changes to cellularphenomena (e.g., cell cycle: phase, pausing, resumption, defects, etc.).

In some embodiments, the present invention provides one or more chemicalprobes comprising, consisting of, or consisting essentially of: A) ametal binding group (e.g., Zn-binding group, general metal chelator,etc.); B) a signaling moiety (e.g., fluorophore) that will exhibit achange in detectable signal (e.g., fluorescence properties) in responseto metal ion binding to the metal binding group; C) a linker group; anattachment group that will allow for attachment of the probe to surfaces(e.g., plate, bead, well, slide, biopolynerm cell surface, etc) viacoupling chemistries (e.g., amide coupling, thiol/maleimide chemistry,click chemistry, etc.).

I. Zn-Responsive Probes

In some embodiments, the present invention provides probes and otherreagents for use in detecting Zn concentrations, levels, presence, orchanges thereof. In some embodiments the present invention providesZn-responsive chemical probes. In some embodiments, the probes andreagents provided herein provide intracellular, extracellular, ornon-cellular detection and/or quantification of Zn, and/or changes inthe presence or concentration of Zn, either in vivo or in vitro. In someembodiments, the probes described herein are capable of (1) detectingthe presence of one or more Zn ions and/or Zn-containing compositions(e.g., by binding to Zn), and (2) signaling the detection of Zn ionsand/or Zn-containing compositions (e.g., optically). In someembodiments, probes comprise one or more of: a Zn-binding group (ZB), asignaling moiety (S) (e.g., fluorophore), one or more linkers (Lx), andan attachment group (A). In some embodiments, a Zn-responsive probecomprises a Zn-binding group attached to a signaling moiety, optionallythrough a linker. In some embodiments, a Zn-binding group and signalingmoiety are directly attached. In some embodiments, a Zn binding groupand/or signaling moiety are covalently connected to an attachment group(e.g., through a linker, directly, etc.). In some embodiments, aZn-responsive probe comprises a general structure selected from thegroup comprising:

(1) A-L-S—ZB

(2) A-L-ZB—S

(3) A-S—ZB

(4) A-S-L-ZB

(5) A-ZB-L-S

(6) A-L1-S-L2 -ZB

(7) A-L1-ZB-L2 -S

(8) S—ZB

In some embodiments, a Zn-responsive probe comprises a Zn-binding groupand a signaling moiety. In some embodiments, the Zn-binding groupcomprises a chemical functionality to bind one or more Zn ions presentin its local environment. In some embodiments, upon binding a Zn ion bythe Zn-binding group, a structural, conformational, chemical, physical,or other change in the Zn-responsive probe causes a detectable change inthe signal from the signaling moiety (e.g., shift in emission maximum,shift in excitation maximum, change in intensity, etc.). In someembodiments, detection or quantification of the signal from thesignaling moiety provides a qualitative and/or quantitative means fordetecting and/or measuring the presence or amount of Zn present in theprobe's local environment. In some embodiments, detection orquantification of changes in the signal from the signaling moietyprovides a qualitative and/or quantitative means for detecting and/ormeasuring changes in the presence or amount of Zn present in the probe'slocal environment.

In some embodiments, a Zn-responsive probe comprises a Zn binding group,and attachment group, and a signaling moiety. In such embodiments, theZn binding group and signaling moiety function to bind Zn and provide asignal indicative of the binding event, as described in the precedingparagraph. In some embodiments, the attachment group is a chemicalmoiety capable of covalently or non-covalently interacting with anotherchemical moiety known as the anchor moiety. In some embodiments,interaction of the attachment group with the anchor moiety results instable attachment of the Zn-responsive probe to the anchor moiety. Insome embodiments an object or surface (e.g., plate, well, bead, slide,etc.) displays one or more anchor moieties. In some embodiments,interaction of the attachment groups of one or more Zn-responsive probeswith one or more anchor groups displayed on a surface results in one ormore Zn-responsive probes being displayed on the surface or object.

In some embodiments, a Zn-responsive probe comprises one or morestructural or functional features described in U.S. Pat. No. 7,105,680;herein incorporated by reference in its entirety.

A. Zn-Binding Groups

In some embodiments, a Zn-responsive probe comprises a metal-bindinggroup. In some embodiments, a metal binding group comprises a Zn-bindinggroup. In some embodiments, a Zn-responsive probe comprises more thanone Zn-binding groups (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). In someembodiments, a Zn-binding group is a chemical moiety capable of stablyinteracting with one or more Zn ions. In some embodiments, a Zn-bindinggroup is capable of interacting with one or more Zn ions, whilecovalently attached to the other functional elements of theZn-responsive probe. In some embodiments, a Zn-binding group interactswith a Zn ion through covalent and/or non-covalent binding. In someembodiments, a Zn-binding group coordinates and/or partially coordinatesa Zn ion. In some embodiments, a Zn-binding group is capable ofcoordinating a single Zn ion. In some embodiments, a Zn-binding group iscapable of coordinating more than one Zn ions at a time (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10 . . . 20 . . . 50 . . . 100 . . . 1000, etc.). In someembodiments, coordination of a Zn ion results in a chemical, magnetic,electric, physical, and/or structural change in the Zn-binding group,the signaling moiety, the connection between the Zn-binding group andthe signaling moiety, and/or the Zn-responsive probe. In someembodiments, the type, degree, and/or magnitude of the change aredependent or responsive to the number of Zn ions coordinated (e.g., morecoordinated Zn ions results in greater change). In some embodiments thedenticity and/or identity of Zn-binding groups is adjusted to tune metalbinding affinity and selectivity.

In some embodiments, the present invention is not limited to anyparticular type or class of Zn-binding groups. In some embodiments, aZn-binding group comprises a functional group capable of transiently orstably binding, coordinating, and/or chelating one or more Zn ions(e.g., free or in another complex). In some embodiments, a Zn-bindinggroup is Zn specific. In some embodiments, a Zn-binding grouppreferentially binds Zn over other metal ions. In some embodiments, aZn-binding group is a general metal-binding moiety. Chemical moietiesthat find use as Zn-binding groups, or within Zn-binding groups, of thepresent invention include, but are not limited to, e.g.,diethyldithiocarbamate (DEDTC) and ethylenediaminetetra-acetic acid(EDTA), 1,10-phenanthroline, pyridyl-containing compounds,amine-containing compounds (e.g., tertiary amines), histidine containingcompounds, sulfonamide-containing compounds, etc.. In some embodiments,a Zn-binding group has at least one functional group selected frompolyalkylene oxide, hydroxylated group, or a group having at least oneamine, ammonium salt, carboxylate, sulfanyl, sulfinyl, sulfonyl,phosphate, phosphonate, phosphate, tertiary amine, pyridyl group; orcombinations thereof. In some embodiments, Zn-binding groups compriseone or more sites for attachment to other functional groups within theZn-responsive probe (e.g., attachment group, signaling moiety, linker,another Zn-binding group, etc.).

B. Signaling Moiety

In some embodiments, a signaling moiety is a detectable chemical moiety.In some embodiments, a signaling moiety is an optically detectablechemical moiety (e.g., fluorophore, chromophore, etc.). In someembodiments, a signaling moiety comprises a fluorescent dye orfluorophore. In some embodiments, a signaling moiety finds utility as afluorophore for detection using one or more of optical spectroscopy,fluorescence spectroscopy, confocal spectroscopy, confocal fluorescencespectroscopy, two-photon excitation (TPE) fluorescence microscopy, etc.

In some embodiments, a signaling moiety is configured within aZn-responsive probe such that coordination of one or more zinc ions bythe Zn-binding group results in a detectable change in the signal fromthe signaling moiety. In some embodiments, a detectable change in signalcomprises a change (e.g., increase or decrease) in signal (e.g.,fluorescence) intensity. In some embodiments a detectable change (e.g.,increase or decrease) in signal (e.g., fluorescence) intensity isreadily detectable by a skilled artisan using the compositions andmethods of the present invention (e.g., 1.1-fold . . . 1.2-fold . . .1.5-fold . . . 2-fold . . . 5-fold . . . 10-fold . . . 20-fold . . .50-fold . . . 100-fold . . . 200-fold . . . 500-fold . . . 1000-fold,etc.). In some embodiments, a detectable change in signal comprises achange (e.g., increase or decrease) in the excitation maximum. In someembodiments, a detectable change in signal comprises a change in theexcitation spectrum. In some embodiments, a detectable change in signalcomprises a change (e.g., increase or decrease) in the emission maximum.In some embodiments, a detectable change in signal comprises a change inthe emission spectrum.

The present invention is not limited to any particular signaling moiety.In some embodiments, the signaling moiety is a fluorophore. The presentinvention is not limited to any particular fluorophore. Fluorophoresand/or fluorescent labels that find use as or within signaling moietiesof the present invention include, but are not limited to, e.g.,fluorescein and rhodamine dyes, such as fluorescein isothiocyanate(FITC), 6-carboxyfluorescein (commonly known by the abbreviations FAMand F), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE or J),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G⁵ or G⁵),6-carboxyrhodamine-6G (R6G⁶ or G⁶), and rhodamine 110; cyanine dyes,e.g., Cy3, Cy5 and Cy7 dyes; alexa dyes, e.g., alexa fluor 555, alexafluor 594; coumarins, e.g., umbelliferone; benzimide dyes, e.g., Hoechst33258; phenanthridine dyes, e.g., Texas Red; ethidium dyes; acridinedyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethinedyes, e.g., cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes andquinoline dyes; and derivities thereof. Suitable fluorescent labelsinclude any of the variety of fluorescent labels disclosed in UnitedStates Patent Application Publication No. 20010009762, the disclosure ofwhich is incorporated herein by reference. In some embodiments,signaling moieties comprise one or more sites for attachment to otherfunctional groups within the Zn-responsive probe (e.g., attachmentgroup, another signaling moiety, linker, Zn-binding group, etc.).

C. Linker

In some embodiments, present invention provides one or more linkers,linking moieties, linking groups, or linker regions. In someembodiments, a linker connects two or more functional groups of aZn-responsive probe (e.g., signaling moiety, Zn-binding group,attachment group, etc.). In some embodiments, a linker comprises 1-1000atoms (e.g., 1-10, 1-100, etc.). In some embodiments, a linker connectsa signaling moiety to a Zn-binding group. In some embodiments, a linkerconnects a signaling moiety to an attachment group. In some embodiments,a linker connects an attachment group to a Zn-binding group. In someembodiments, one functional groups of a Zn-responsive probe (e.g.,signaling moiety, Zn-binding group, attachment group, etc.) is connectedto more than one other functional groups of a Zn-responsive probe (e.g.,signaling moiety, Zn-binding group, attachment group, etc.) by multiplelinkers. In some embodiments, a linker is branched for connection ofthree or more functional groups of a Zn-responsive probe (e.g.,signaling moiety, Zn-binding group, attachment group, etc.).

The present invention is not limited to any particular linker group.Indeed, a variety of linker groups are contemplated, suitable linkerscould comprise, but are not limited to, alkyl groups, ether, polyether,alkyl amide linker, a peptide linker, a modified peptide linker, aPoly(ethylene glycol) (PEG) linker, a streptavidin-biotin oravidin-biotin linker, polyaminoacids (e.g. polylysine), functionalizedPEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93/06868and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990),herein incorporated by reference in their entireties), PEG-chelantpolymers (WO94/08629, WO94/09056 and WO96/26754, herein incorporated byreference in their entireties), oligonucleotide linker, phospholipidderivatives, alkenyl chains, alkynyl chains, disulfide, or a combinationthereof.

In some embodiments the linker comprises a single chain connecting onefunctional groups of a Zn-responsive probe (e.g., signaling moiety,Zn-binding group, attachment group, etc.) to another functional group ofa Zn-responsive probe (e.g., signaling moiety, Zn-binding group,attachment group, etc.). In some embodiments, there are multiple linkersconnecting multiple Zn-binding groups to a single signaling moiety. Insome embodiments, a linker may connect multiple Zn-binding groups toeach other. In some embodiments, a linker may connect multiple signalingmoieties to each other. In some embodiments, a linker may be branched.In some embodiments, the linker may be flexible, or rigid.

In some embodiments, the linker of the present invention is cleavable orselectively cleavable. In some embodiments, the linker is cleavableunder at least one set of conditions, while not being substantiallycleaved (e.g. approximately 50%, 60%, 70%, 80%, 90%, 95%, 99%, orgreater remains uncleaved) under another set (or other sets) ofconditions. In some embodiments, the linker is susceptible to cleavageunder specific conditions relating to pH, temperature, oxidation,reduction, UV exposure, exposure to radical oxygen species, chemicalexposure, light exposure (e.g. photo-cleavable), etc. In someembodiments, the linker region is photocleavable. That is, upon exposureto a certain wavelength of light, the linker region is cleaved, allowingrelease of the connected functional groups (e.g., Zn-binding group,etc.). This embodiment has particular use in developmental biologyfields (cell maturation, neuronal development, etc.), where the abilityto follow the fates of particular cells is desirable. In someembodiments, a preferred class of photocleavable linkers are theO-nitrobenzylic compounds, which can be synthetically incorporated viaan ether, thioether, ester (including phosphate esters), amine orsimilar linkage to a heteroatom (particularly oxygen, nitrogen orsulfur). Also of use are benzoin-based photocleavable linkers. A widevariety of suitable photocleavable moieties is outlined in the MolecularProbes Catalog, supra. In some embodiments, the linker is susceptible toenzymatic cleavage (e.g. proteolysis). In some embodiments of thepresent invention, functional groups (e.g., signaling moiety andZn-binding group) are linked, via a cleavable linker. The presentinvention is not limited to any particular linker group. In someembodiments, the cleavable linker region contains a peptide portion. Insome embodiments, the peptide portion of the cleavable linker region iscleavable. In some embodiments, the peptide portion of the cleavablelinker region is enzymatically cleavable. In some embodiments thecleavable linker contains a specific proteolytic site. In someembodiments, in addition to the peptide portion of the cleavable linkerregion, an additional linker portion is contemplated. Indeed, a varietyof additional linker groups are contemplated, suitable linkers couldcomprise, but are not limited to, alkyl groups, ether, polyether, alkylamide linker, a peptide linker, a modified peptide linker, aPoly(ethylene glycol) (PEG) linker, a streptavidin-biotin oravidin-biotin linker, polyaminoacids (e.g. polylysine), functionalisedPEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93/06868and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990),herein incorporated by reference in their entireties), PEG-chelantpolymers (WO94/08629, WO94/09056 and WO96/26754, herein incorporated byreference in their entireties), oligonucleotide linker, phospholipidderivatives, alkenyl chains, alkynyl chains, disulfide, or a combinationthereof.

D. Attachment Group

In some embodiments, an attachment group is configured for covalentand/or non-covalent binding/interaction with a surface (e.g., well,plate, bead, slide, etc.). In some embodiments, an attachment group isconfigured for covalent and/or non-covalent binding/interaction with ananchor moiety. In some embodiments, an attachment group and anchormoiety comprise an attachment pair. In some embodiments, an attachmentgroup and anchor moiety are chemically or physically complimentary toallow a stable interaction (e.g., covalent or non-covalent) betweenthem. In some embodiments, an attachment group is part of aZn-responsive probe. In some embodiments, an anchor moiety is afunctional group attached to a surface or object (e.g., plate, bead,well, slide, etc.). In some embodiments, the interaction of the membersof an attachment pair provides a mean for attaching a Zn-responsiveprobe to a surface or object (e.g., plate, bead, well, slide, etc.). Insome embodiments, the attachment pair interaction is reversible. In someembodiments, the attachment pair interaction is irreversible. In someembodiments, the interaction of the attachment pair is stable enough toprovide stable attachment of the Zn-responsive element to a surface orobject (e.g., plate, bead, well, slide, etc.) for at least the durationof Zn detection (e.g., experiment, evaluation, assay, treatment,diagnosis, etc.). The present invention is not limited by the type orclass of attachment group. In some embodiments, attachment groupscomprise, e.g., one or more chemical groups including, but not limitedto: aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate,tresylate, azirdine, oxirane, orthopyridyl disulphide, vinylsulfone,iodoacetamide or a maleimide, a group suitable for Click chemistry, etc.

In some embodiments, an attachment pair (e.g., attachment group andanchor moiety) provide chemical means for attaching a Zn-responsiveprobe to a surface (e.g., plate, bead, well, slide, etc.). The presentinvention is not limited by the types of attachment pairs, and anysuitable attachment pair that allows for attachment of a Zn-responsiveprobe to an anchor-decorated surface may find use with the presentinvention (e.g., carboxylate and amine; thiol and maleimide; biotin andstreptavidin; azide and phosphine, etc.).

In some embodiments, an attachment pair (e.g., attachment group andanchor moiety) provide chemical means for attaching a Zn-responsiveprobe to a surface (e.g., plate, bead, well, slide, etc.). The presentinvention is not limited by the types of surfaces, and any suitablesurface or object may find use with the present invention, for example:beads, nanobeads, microparticles, microscope slide, microfluidicschamber, the interior of a reaction tube, a well (e.g., of a 96-wellplate, of a 384-well plate, etc.), microtiter plate, etc.

In some embodiments, attachment of one or more Zn-responsive probes to asurface allows a sample to be added to the Zn-responsive probes, assayedfor the presence, amount, or a change in Zn concentration, and then thesample is removed from the surface without contaminating the sample. Insome embodiments, non-invasive sample assaying is provided by stablyanchored Zn-responsive probes. In some embodiments, attachment of one ormore Zn-responsive probes to a bead allows the Zn-responsive probes tobe added to a sample; the sample is assayed for the presence, amount, ora change in Zn concentration; and then the bead-bound Zn-responsiveprobes are removed from the sample without contamination. In someembodiments, attachment of one or more Zn-responsive probes to a surfaceallows a sample to be added to the Zn-responsive probes withoutcontamination of the sample with the probes. In some embodiments,attachment of one or more Zn-responsive probes to a surface allows cells(e.g., oocytes) to be added to the Zn-responsive probes without entry ofthe probes into the cells (e.g., preventing cell toxicity or disruptionof cellular functions).

II. Methods/Applications

In some embodiments, the present invention provides methods fordetection, measurement, identification, and/or quantification of Zn ionsand/or Zn-containing compounds or compositions. In some embodiments, thepresent invention provides methods for detection, measurement,identification, and/or quantification of Zn concentration. In someembodiments, the present invention provides methods for detection,measurement, identification, and/or quantification of changes in Znconcentration. In some embodiments, the present invention providesmethods for correlating biological and/or cellular events, changes,processes, and/or phenomena to Zn concentration or changes thereof(e.g., extracellular Zn concentration, intracellular Zn concentration,etc.). In some embodiments, any suitable methods of detecting and/orquantifying the presence and/or concentration of Zn ions, or changesthereof, find use in the present invention. In some embodiments, Zn ionsare detected and or quantified through the use of Zn-sensitive probes,e.g., those described herein.

In some embodiments, the present invention provides detection orquantification of changes in intracellular and/or extracellular Znconcentration and correlates those changes to changes in the cell cycle(e.g., stall, pause, arrest, resumption). In some embodiments, thecompositions and methods herein provide correlation of Zn concentration(e.g., extracellular Zn concentration) to the cell cycle phase of anoocyte (e.g., maturing oocyte) and/or is progression through thematuration process. In some embodiments, methods provided herein detectand/or quantify Zn uptake or release from cells. In some embodiments,methods correlate Zn uptake or release from cells with cellular activity(e.g., cell cycle activities (e.g., moving from one phase to another,arrest of cell cycle, resumption of cell cycle)).

Experiments were conducted during development of the present inventionto determine whether zinc regulation of meiotic progression extended tocell cycle regulation in the fertilized egg. The dynamics of zinc andcalcium within the physiological context of fertilization were examinedusing a variety of chemical probes, which revealed that calciumoscillations induced a rapid loss of zinc through an event termed thezinc spark. Experiments conducted during development of the presentinvention demonstrated that zinc sparks (i.e., Zn release from cells(e.g., oocytes) are evolutionarily conserved in three mammalian species,and that these fluxes in intracellular zinc availability mediate cellcycle resumption. Experiments conducted during development of thepresent invention demonstrate a zinc-dependent mechanism for cell cycleregulation in the mammalian egg. Calcium oscillations initiate and arerequired for the programmed loss of cellular zinc via the zinc sparksand that in turn drives cell cycle resumption.

Experiments conducted during development of embodiments of the presentinvention demonstrate that intracellular Zn levels in maturing oocytesrise dramatically as the cell matures from prophase-I to metaphase-II.This rise in Zn levels corresponds to uptake in Zn ions from theextracellular environment. Cells that do not undergo this Zn acquisitiondo not progress to metaphase-II arrest, and are not candidates forfertilization. Cells that do not acquire the requisite intracellular Znion concentration proceed to telophase-I arrest and are of poor qualityfor fertilization (e.g., unlikely to be successfully fertilized).Detection of the uptake of Zn ions by oocytes maturing from prophase-Ito metaphase-II provides a marker for healthy, normal maturation ofoocytes. Detection of the uptake of Zn ions by oocytes maturing fromprophase-I to metaphase-II provides a marker for oocytes that are goodcandidates for fertilization (e.g., via in vitro fertilization (IVF),via in vitro maturation (IVM), etc.). In some embodiments, the presentinvention provides compositions (e.g., Zn-responsive probes) and methodsfor the detection of uptake of Zn ions by cells (e.g., oocytes) from theextracellular environment. In some embodiments, the present inventionprovides compositions (e.g., Zn-responsive probes) and methods forcorrelating the uptake of Zn ions by cells (e.g., oocytes) from theextracellular environment with cellular phenomena (e.g., progress fromprophase-I to metaphase-II).

Further experiments demonstrated that one or more rapid decreases inintracellular Zn concentration occur following fertilization of ametaphase-II oocyte. These decreases in intracellular Zn levelscorrespond to rapid releases of Zn (e.g., Zn sparks) from oocytes intothe extracellular environment. Upon successful fertilization, oocytesexhibit one or more Zn sparks (e.g., 2-7, 3-5, 1-10, at least 3, atleast 4, at least 5, etc.). Detection of the release of Zn ions fromoocytes following fertilization (e.g., Zn sparks) provides a marker forhealthy, normal maturation of oocytes from metaphase-II arrest to atwo-cell embryo. Detection of the Zn ion release (e.g., Zn sparks) byoocytes provides a marker for oocytes that that have been successfullyfertilized (e.g., by IVF). In some embodiments, the present inventionprovides compositions (e.g., Zn-responsive probes) and methods for thedetection of the release of Zn ions from cells (e.g., oocytes) into theextracellular environment. In some embodiments, the present inventionprovides compositions (e.g., Zn-responsive probes) and methods forcorrelating the release of Zn ions from cells (e.g., oocytes) into theextracellular environment with cellular phenomena (e.g., fertilization).

In some embodiments, the present invention provides compositions andmethods for the detection of Zn release and/or uptake from cells, andcorrelation thereof to cellular phenomena. In some embodiments, suchdetection and correlation find use as a marker of cellular phenomenaand/or cellular progression. In some embodiments, provided are methodsfor the non-invasive selection of quality eggs (e.g., high quality,e.g., matured to metaphase-II) for human IVF. In some embodiments,provided are methods for non-invasive selection of high quality eggs(e.g., matured to metaphase-II) for domestic animal and/or companionanimal IVF. In some embodiments, provided are methods for non-invasiveselection of high quality eggs (e.g., from any species) for use in thederivation of stem cells. In some embodiments, provided herein arefunctional marker for progression of egg (e.g., human, non-human) in IVM(e.g., with the purpose of freezing only good quality eggs).

The compositions and methods of the present invention are not limited toapplications regarding oocyte cell cycle (e.g. detection of: cell cyclephase, stalling, pausing resumption, etc.). In some embodiments, thecompositions and methods herein find utility in any cell types (e.g.,oocyte, sperm cell, islets, neurons, etc.), as well as in cell lysate ornon-cellular conditions.

In some embodiments, zinc concentration and/or changes in zincconcentration are detected in pancreatic islet cells (e.g., to identifyislet cells capable of reacting properly to glucose concentration, toidentify islet cells capable of appropriately releasing insulin, etc.).In some embodiments, zinc concentration and/or changes in zincconcentration are detected in pancreatic islet cells to evaluate thequality of the cells for potential use in islet cell transplant therapy.In some embodiments, the present invention provides methods formonitoring islet cell activation.

In some embodiments, zinc concentration and/or changes in zincconcentration are detected in sperm cells to evaluate sperm quality(e.g., for fertility treatment and/or evaluation).

In some embodiments, zinc concentration and/or changes in zincconcentration are detected in neuronal cells (e.g., to detect proper Znrelease, to detect diseases of the nervous system (e.g., Parkinson's,Alzheimer's, etc.). In some embodiments, the present invention providesmethods for monitoring neuron development. In some embodiments, zincconcentration and/or changes in zinc concentration are detected inneural cells to evaluate cell quality (e.g., appropriate levels andtiming of metal (e.g., Zn) release) for neural cell transplanttherapies.

In some embodiments, the present invention is not limited to detectionor cellular, intracellular, or extracellular zinc. In some embodiments,methods and compositions herein are suitable for detection of zinc inany sample (e.g., biological (e.g., blood, urine, saliva, etc.),environmental (e.g., soil, water, contaminant, waste, etc.), veterinary,clinical, etc.)

Experimental EXAMPLE 1 Materials and Methods

Mouse egg collection. Eggs were collected from adult (6-8 weeks old)female mice of the CD-1 strain. Micewere super stimulated with an i.p.injection of 5 IU pregnant mare's serum gonadotropin (PMSG, EMDBiosciences, San Diego, Calif.), followed 46-48 h later by an i.p.injection of 5 IU human chorionic gonadotropin (hCG, Sigma-Aldrich, St.Louis, M.). Mice were sacrificed by CO2 asphyxiation and cervicaldislocation 13-14 h post-hCG administration. Clutches of cumulus-oocytecomplexes were isolated from the oviducts. Cumulus cells were denudedusing 30 μg/ml hyaluronidase and gentle aspiration through a narrow-borepipette. Animals were treated in accord with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals. Food and waterwere given ad libitum. The Northwestern University Institutional AnimalCare and Use Committee (IACUC) approved all protocols.

Time-lapse imaging of mouse eggs during in vitro fertilization. Spermfrom the cauda epididymides of proven breeder CD-1 males were used forsperm in vitro fertilization (IVF). The sperm population was purifiedusing Percoll gradient centrifugation (PGC) as described previously (Xuet al. (2006), Biol Reprod 75, 916-23.; herein incorporated by referencein its entirety)) and capacitated for up to 3 h in IVF medium composedof potassium simplex optimized medium (KSOM, Millipore, Billerica,Mass.) supplemented with 3 mg/ml bovine serum albumin (BSA, MPBiomedicals, Solon, Ohio) and 5.36 mM D-glucose (Sigma-Aldrich).Superovulated eggs were collected as described above. The zona pellucida(ZP) was removed by brief treatment in acidic Tyrode's solution (pH 2.5,Millipore). The ZP-free eggs were allowed to settle in a 50 μl drop ofcalcium- and magnesium free Dulbecco's PBS (DPBS, Invitrogen, Carlsbad,Calif.) on a glass-bottom dish (Bioptechs Inc., Butler, Pa.) coated withpoly-L-lysine. After 10 min, IVF medium containing 20 μM FluoZin-3(Invitrogen) was slowly added to a final volume of 1 ml, taking care notto disturb the eggs. The final DMSO concentration was 0.1% (v/v), ashigher concentrations were cytotoxic to sperm. Capacitated sperm wereadded to the eggs to a final concentration of 1.0×10⁶ sperm/ml and imageacquisition began immediately upon addition of sperm. Images wereacquired every 4 sec for a total duration of 3 hours. All images wereacquired on a TCS SP5 confocal microscope (Leica Microsystems,Heidelberg, Germany) equipped with a stage top incubator (Tokai Hit,Shizuoka, Japan), 20× objective, and an Ar (488 nm) laser line.

Parthenogenetic activation and pyrithione treatment of mouse eggs.Strontium chloride (SrC12) was prepared as a 1 M stock solution incalcium-free KSOM (Millipore) and stored as ready-to-use aliquots at−20° C. SrC12 was diluted to a working concentration of 10 mM incalcium-free KSOM. Eggs were incubated in SrC12 for 90 min. In somecases, eggs were then treated with zinc pyrithione (ZnPT,Sigma-Aldrich). Dose response experiments were performed to testcytotoxicity; a distinct cytotoxic effect was seen at concentrationsabove 20 μM. Therefore, ZnPT concentrations were reduced to 10 μM andtreatment time was limited to 10 min at 37° C. in an atmosphere of 5%CO2. Both control and ZnPT-treated eggs were washed and transferred tofresh KSOM for extended culture.

Non-human primate oocyte collection, in vitro maturation, andparthenogenetic activation. Ovarian tissue was surgically dissected fromMacaca mulatta (rhesus macaque, 3 years old) and Macaca fascicularis(crab-eating macaque, 3 years old) females at the Panther TracksLearning Center (Immokalee, Fla.). The tissue was cut in half, placed in15 ml of SAGE oocyte washing medium (Cooper Surgical, Trombull, Conn.),and shipped overnight at ambient temperature. The tissue was processedwithin 18 hours of surgery. Large follicles were opened by grazing withan insulin-gauge needle. Oocytes of diameter greater than 100 μm(including the zona pellucida) were collected for in vitro maturation(IVM), whether they were enclosed by cumulus cells or denuded. They wereplaced in an IVM medium composed of SAGE oocyte maturation medium(Cooper Surgical) supplemented with 75 mIU/μl follicle stimulatinghormone (FSH, a gift from Organon, Roseland, N.J.), 75 mIU/μlluteinizing hormone (LH, a gift from Ares Serono, Randolph, Mass.), 1.5IU/μl hCG (Sigma-Aldrich), and 5 ng/ml epidermal growth factor (EGF, BDBiosciences, San Jose, Calif.). Oocytes were observed up to 48 hourspost-maturation and used immediately for imaging upon complete extrusionof a polar body.

Time-lapse imaging of parthenogenetically activated eggs. Eggs werefirst incubated in 10 μM Calcium Green-1 AM (Invitrogen) and 0.04% (w/v)Pluronic F-127 (Invitrogen) for either 30 min (mouse) or 60 min(non-human primate) at 37° C. Mouse eggs were washed in calcium-freeKSOM (Millipore) and transferred to a 50 μl drop containing 10 mM SrC12(Sigma-Aldrich) and 50 μM FluoZin-3 (Invitrogen) on a glass-bottom dish(Bioptechs Inc.). Non-human primate eggs were transferred to one end ofa long and shallow drop (20 μl) of calcium-free KSOM (Millipore)containing 50 μM FluoZin-3 (Invitrogen) on a glass-bottom dish(Bioptechs Inc.), covered with oil for embryo culture (IrvineScientific, Santa Ana, Calif.). Ionomycin was perfused in at theopposite end to a final concentration of 20 μM. In both cases, imageacquisition began immediately and occurred every 4 s for a total of 1-3hours (mouse) or 15 min (non-human primate), unless noted otherwise. Allimages were acquired on a TCS SP5 confocal microscope (LeicaMicrosystems) equipped with a stage top incubator (Tokai Hit), 20×objective, and an Ar (488 nm) laser line.

Imaging of total and labile zinc in the mouse egg. Fertilized eggs werecollected at two and six hours post-fertilization were preparedwhole-mount for synchrotron-based x-ray fluorescence microscopy (XFM).Cells were transferred with a minimal amount of media to an intact 5mm×5 mm silicon nitride window (Silson, Blisworth, U.K.) on a heatedstage warmed to 37° C. When most of the media had evaporated withoutdrying out the sample, 1 μl of ammonium acetate solution (100 mM, 4° C.)was administered to each sample under a dissection microscope. Thisfacilitated a quick wash and dehydration process, leaving the morphologyof the sample intact without causing membrane rupture. XFM was performedat Beamline 2-ID-E at the Advanced Photon Source (Argonne NationalLaboratory, Argonne, Ill.). 10 keV x-rays were monochromatized with asingle bounce Si(111) monochromator, and focused to a spot size of0.5×0.6 μm using Fresnel zone plate optics (X-radia, Concord, Calif.).Raster scans were done in steps of 1 μm. Fluorescence spectra werecollected with a 1 sec dwell time using a silicon drift detector(Vortex-EM, SII NanoTechnology, CA). Quantification and image processingwas performed with MAPS software. The fluorescence signal was convertedto a two-dimensional concentration in μg/cm2 by fitting the spectraagainst the thin-film standards NB S-1832 and NBS-1833 (National Bureauof Standards). It was assumed that no elemental content was lost duringsample preparation. The data was compared to the total zinc content inunfertilized eggs and two-cell embryos. The data for the two-cell embryowas already published in a previous report14. The distribution of labilezinc was interrogated in live, unfertilized mouse eggs using twoindependent zinc fluorophores, zinquin ethyl ester (Sigma-Aldrich) andFluoZin-3 AM (Invitrogen). Eggs were incubated in either 20 μM zinquinethyl ester in combination with 1 μM Syto 64 (Invitrogen) or 10 μMFluoZin-3 AM in combination with 10 μg/ml Hoechst 33342 (Invitrogen) for60 min in KSOM (Millipore). They were imaged in 20 μl drops of KSOMcovered with oil for embryo culture (Irvine Scientific) on glass-bottomdishes (Bioptechs Inc.). Images were acquired as Zstacks on a TCS SP5confocal microscope (Leica Microsystems) equipped with a stage topincubator (Tokai Hit), 63× oil-immersion objective, and HeNe (543 nm),Ar (488 nm), and near-UV (405 nm) laser lines.

TPEN treatment and spindle imaging of mouse eggs. Superovulated eggswere collected and transferred to KSOM medium (Millipore) with orwithout 10 μM TPEN and cultured up to 8 hrs at 37° C. in an atmosphereof 5% CO2 in air. Previous work showed that treatment of maturingoocytes with 10 μM TPEN could selectively limit the intracellularacquisition of zinc (relative to iron and copper) but withoutsignificant toxicity14. Eggs were fixed and permeabilized for 30 min at37° C. in a solution containing 2% formaldehyde, 2% Triton X-100, 100 mMPIPES, 5 mM MgCl2, and 2.4 mM EGTA. Eggs were then washed and blockedfor 1-3 hrs in 1× PBS containing 0.1 M glycine, 3 mg/mL BSA, 0.01%Tween-20, and 0.01% sodium azide followed by incubation with anti-<-tubulin (1:100, Sigma) in blocking buffer for 1 hr at 37° C. Eggs werewashed again in blocking buffer, incubated in Alexa Fluor 488-conjugatedgoat anti-mouse IgG (Invitrogen) and rhodamine-phalloidin conjugate(Invitrogen) at 37° C. for 1 hr, washed three additional times, andmounted on microscopy slides with coverslips in Vectashield with DAPI.Images were acquired as Z-stacks on a TCS SP5 confocal microscope (LeicaMicrosystems) equipped with a stage top incubator (Tokai Hit), 63×oil-immersion objective, and HeNe (543 nm), Ar (488 nm), and near-UV(405 nm) laser lines.

EXAMPLE 2 Release of Zinc at Egg Activation is a Conserved Event inMammalian Species

In experiments conducted during development of embodiments of thepresent invention extracellular zinc events were monitored duringfertilization of mouse eggs using a membrane impermeant derivative ofthe zinc fluorophore FluoZin-3 (Qian & Kennedy. (2002) J Am Chem Soc124, 776-8.; herein incorporated by reference in its entirety).Strikingly, fertilization triggered the repetitive release of zinc intothe extracellular milieu (SEE FIG. 1A and 1B). These events were termedzinc sparks for their brevity and intensity. Successful fertilizationwas confirmed by extrusion of the second polar body (SEE FIG. 1C and 1D,PB2). These events were recapitulated during parthenogenesis of mouseeggs (SEE FIG. 1E). Zinc sparks are an evolutionarily conservedphenomenon, as they were also observed during parthenogenetic activationof two different non-human primate species, Macaca mulatta (rhesusmacaque) and Macaca fascicularis (crab-eating macaque) (SEE FIG. 2).Additional characterization of the zinc sparks was completed usingparthenogenetically activated mouse eggs.

The dynamics of the zinc sparks were variable from egg to egg. Zincsparks occurred during the first 90 min of activation, and individualeggs exhibited between one and five zinc sparks, which were counted in aplot of fluorescence intensity over time (SEE FIG. 1A). Eighty-onepercent of all eggs examined exhibited two or three exocytosis events,while eleven percent exhibited one event and eight percent exhibitedfour or five events. No more than five zinc sparks were observed in anyof the samples. The interval between each exocytic event was 9.5±0.8 min(mean±SEM). The range of intervals was between 3.46 and 26.00 min.

Zinc sparks could be induced by fertilization or by parthenogenesisusing two different reagents: strontium chloride in rodents andionomycin in two non-human primate species. While strontium chlorideinduces multiple calcium transients whereas ionomycin only induces asingle calcium transient (Kline & Kline. (1992) Dev Biol 149, 80-9.;Markoulaki et al. (2003) Dev Biol 258, 464-74.; herein incorporated byreference in their entireties). Accordingly, multiple zinc sparks wereobserved in rodents whereas only a single zinc spark was seen in thenon-human primate species. This result indicates calcium dependence ofthe zinc sparks.

EXAMPLE 3 Initiation of the Zinc Sparks is Dependent on IntracellularCalcium Transients

Experiments were conducted during development of embodiments of thepresent invention to determine the role of zinc sparks within thetemporal context of the calcium oscillations. Intracellular calciumoscillations were simultaneously monitored alongside zinc sparks usingthe calcium fluorophore Calcium Green-1 AM (ex. 506, em. 531), which hassimilar excitation and emission spectra to FluoZin-3 (SEE FIG. 3A). Eachzinc spark was closely associated with an intracellular calciumtransient (SEE FIG. 3B); upon closer inspection, it was found that eachzinc spark was immediately preceded by an elevation in intracellularcalcium (SEE FIG. 3C). Parallel experiments employing a membranepermeable zinc fluorophore, FluoZin-3 AM, did not reveal clearintracellular zinc transients (SEE FIG. 4A), ruling out the possibilitythat large oscillations in intracellular free zinc contributes to theintracellular transients detected by Calcium Green-1 AM. It wasconfirmed that this was representative of successfully activated eggs bysimultaneously monitoring second polar body (PB2) extrusion bybrightfield imaging (SEE FIG. 4A). Further control experiments employingone fluorophore at a time reveal that the zinc sparks are not dependentupon or effected by the presence of the calcium probe; for example,detection of zinc sparks using FluoZin-3 in the absence of CalciumGreen-1 AM (SEE FIG. 1A (fertilization) and FIG. 1E (parthenogenesis)).

The observation that calcium transients immediately preceded each zincrelease event indicates that the zinc sparks are directly dependent onintracellular calcium fluxes. Additional experiments were consistentwith this determination. First, Zinc sparks did not occur in the absenceof an activating agent and hence, a lack of calcium oscillations (SEEFIG. 4B). Second, zinc sparks were absent in eggs treated with themembrane permeable acetoxymethyl ester (AM) derivative of1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Thisreagent is commonly used to suppress calcium oscillations in eggs (Kline& Kline. (1992) Dev Biol 149, 80-9.; Tahara et al. (1996) Am J Physiol270, C1354-61.; herein incorporated by reference in their entireties)and as such, both calcium transients and zinc sparks were absent inBAPTA AM-treated eggs even after extended culture in strontium chloride(SEE FIG. 4C). Furthermore, these eggs did not extrude a second polarbody or form a pronucleus (SEE FI. 4D and 4E). Taken together, thesedata indicate that the zinc sparks are preceded by and depend upon thecalcium oscillations that begin early in the egg activation process.

EXAMPLE 4 Localization of Zinc is Cortically Polarized in Mature Eggs

Zinc sparks were excluded from the region corresponding to the locationof the meiotic spindle, as confirmed by the extrusion of the secondpolar body at that site (SEE FIG. 3A, arrowheads). To investigatepossible cellular sources of the zinc release, the distribution of zincwas determined in unfertilized eggs by synchrotron-based x-rayfluorescence microscopy (XFM). This technique provides the localizationand abundance of the total zinc content of a sample, including both theloosely bound (labile or chelatable) and tightly bound cellular pools,and was previously used to quantify zinc and other transition metals inmaturing oocytes (Kim et al. (2010) Nat Chem Biol 6, 674-81.; hereinincorporated by reference in its entirety). The XFM images revealeddistinct regions of high zinc concentration that formed a polarized andhemispherical pattern (SEE FIG. 5A; i-iii represent three independentsamples). Other essential transition metals such as iron exhibit ahomogeneous distribution across the entire cell (SEE FIG. 3B; i-iiirepresent three independent samples). Quantitative analysis revealedthat zinc was an order of magnitude more abundant than either iron orcopper at all points between the mature, in vivo ovulated (IVO) MIIstage egg and the two-cell embryo (24 hours post-fertilization, or hpf).There is a downward trend with a mean loss of about six billion ions ofzinc upon fertilization, and this trend continued at 6 hpf and 24 hpf(SEE FIG. 6). ANOVA analysis noted a significant difference in the meanzinc content across the four timepoints. Fluorescence imaging ofintracellular zinc confirmed that the amount of chelatable zinc alsodecreased in activated eggs following the zinc sparks (SEE FIG. 7).

Experiments were conducted during development of embodiments of thepresent invention to determine whether the polarized pattern for totalzinc correlated with the distribution of labile (or chelator-accessible)zinc in the egg. Two chemically distinct, zinc-selective fluorophoreswere used to visualize loosely bound zinc in live, unfertilized eggs.Projected images of confocal Z-stack series revealed that zincfluorescence was concentrated away from the meiotic spindle, which wasmarked with fluorescent DNA probes (SEE FIG. 5C-5H). This was alsoevident when analyzing the Z-series slice by slice, which clearlyillustrated higher cortical localization of zinc relative to thecytoplasm (SEE FIG. 8). Furthermore, zinc was present in distinctintracellular compartments as indicated by punctate foci offluorescence. This pattern was independent of the molecular propertiesof the zinc probe: both zinquin ethyl ester (SEE FIG. 5C) and FluoZin-3AM (SEE FIG. 5F) yielded the same general pattern wherein a majority ofthe foci have a peripheral localization. These probes have pM values of9.3 and 8.8, respectively, where pM (like pH) reflects the log of thefree ion concentration (Qian & Kennedy. (2002) J Am Chem Soc 124,776-8.; Fahrni & O'Halloran. (1999) J Am Chem Soc 121, 11448-11458.;herein incorporated by reference in their entireties). Given that the pMvalue of zinc-binding metalloproteins are significantly higher, forexample, carbonic anhydrase has a pM value of 12.4 (Fahrni & O'Halloran.(1999) J Am Chem Soc 121, 11448-11458.; herein incorporated by referencein its entirety), it is unlikely that the probes are detecting zinctightly bound to metalloproteins. Rather, the probes are delineatingcompartments with elevated concentrations of free or weakly bound zincions. In addition to their punctate distribution, the pools ofchelator-accessible zinc were concentrated towards the vegetal pole andaway from the meiotic spindle, whose location was marked with live-cellnuclear stains Syto 64 (SEE FIG. 5D) or Hoechst 33342 (SEE FIG. 5G) asseen in the merged images (SEE FIG. 5E and 5H). The polarizeddistribution detected both by the zinc fluorophores and by XFMcorroborates a significant cortical compartmentalization of zinc in theegg. The polarized distribution of labile zinc mirrors the polarizedrelease of zinc during the zinc sparks, indicating that some fraction ofthese zinc-enriched compartments act the source of the coordinated zincrelease events, i.e., the zinc sparks.

The polarization of the zinc-enriched vesicles mirrors that of thecortical granules (CG), which are exocytic vesicles that participate inthe cortical reaction, which involves the hardening of the zonapellucida to establish a block to polyspermy at fertilization(Ducibella. (1996) Hum Reprod Update 2, 29-42.; herein incorporated byreference in its entirety). To inhibit exocytosis, eggs were treatedwith cytochalasin B (CytoB), an agent that disrupts actin reorganizationand has previously been shown to disrupt CG exocytosis in mouse andhamster eggs (Tahara et al. (1996) Am J Physiol 270, C1354-61.; Zhang etal. (2005) Hum Reprod 20, 3053-61.; incorporated herein by reference inits entirety). CytoB-treated eggs still undergo normal calciumoscillations upon activation but only exhibit a single zinc sparkconcomitantly with the first calcium transient (SEE FIG. 9). In mostcases, subsequent sparks were inhibited (SEE FIG. 9). Experimentsconducted during development of embodiments of the present inventionindicate that zinc-containing vesicles are pre-fused with the eggmembrane, allowing the first spark to occur even in the presence ofCytoB. This type of phenomenon has been described for cortical granulesin the sea urchin egg, where one subset of CGs are in a hemifused stateprior to activation21.

To determine whether the zinc sparks participate in zona hardening likethe cortical reaction, a zinc-insufficient egg model that was obtainedby treating oocytes with the heavy metal chelator TPEN during in vitromaturation was used (Kim et al. (2010) Nat Chem Biol 6, 674-81.; hereinincorporated by reference in its entirety). These eggs did not undergozinc sparks, even when activated in the same dish as control eggs.Zinc-insufficient eggs fertilize at rates comparable to control eggs.Additionally, zinc-insufficient eggs form the normal number ofpronuclei, as indicated by the presence of only one male and one femalepronucleus (Kim et al. (2010) Nat Chem Biol 6, 674-81.; hereinincorporated by reference in its entirety). Therefore, there is noevidence to suggest that the zinc sparks participate in the block topolyspermy.

EXAMPLE 5 Meiotic Resumption at Egg Activation is Dependent on the ZincSparks

The zinc-insufficient egg model revealed a critical function for zinc inthe proper establishment of cell cycle arrest in the mature egg (Kim etal. (2010) Nat Chem Biol 6, 674-81.; herein incorporated by reference inits entirety), which was supported by the observation that chelation ofzinc initiated egg activation (Suzuki et al. (2010) Development 137,2659-2669; herein incorporated by reference in its entirety).Experiments were conducted during development of embodiments of thepresent invention to determine whether a decrease in bioavailable zincwas necessary to permit downstream developmental events upon eggactivation. To address this possibility, activated eggs were treatedwith zinc pyrithione (ZnPT), a zinc ionophore that elevates theintracellular free zinc content of mammalian cells (Taki. (2004) J AmChem Soc 126, 712-3.; herein incorporated by reference in its entirety).First, a titration was performed on unfertilized eggs and did notobserve cytotoxic effects until concentrations of ZnPT reached 50 μM(SEE FIG. 10). To minimize such cytotoxic effects, a fivefold lowerconcentration of ZnPT (10 μM) was used for subsequent experiments. Eggswere subjected to a 10 min ZnPT treatment after the completion of thezinc sparks at 1.5 hours postactivation (hpa) (SEE FIG. 11A). Control(untreated) and ZnPT-treated eggs were then cultured until pronuclearformation was observed in the control group (6-8 hpa; SEE FIG. 11B and11C). Ninety percent of control eggs formed an organized pronucleus thatwas visible by brightfield (SEE FIG. 11B, arrowheads) and also byfluorescence (SEE FIG. 11D). F-actin was homogeneous around the cell(SEE FIG. 11E) and a-tubulin was compact in a spindle remnant (SEE FIG.11F) between the egg and the second polar body (SEE FIG. 11G).

In contrast, only seventeen percent of ZnPT-treated eggs formed apronucleus; the majority did not have a pronuclear structure that wasclearly visible by brightfield (SEE FIG. 11C). In eighty-four percent ofthese eggs lacking a pronucleus, we noted the presence of alignedchromosomes (SEE FIG. 11H), instead of the membrane-enclosed nucleusobserved in the control eggs.

Notably, an actin-enriched region in the ZnPT-treated eggs was alsoobserved (SEE FIG. 11I, arrow) at the cortical region overlying thechromosomes (SEE FIG. 11J). This “actin cap” is a polarized featurethought to be unique to unfertilized eggs (Longo & Chen. (1985) Dev Biol107, 382-94.; Duncan et al. (2005) Dev Biol 280, 38-47.; hereinincorporated by reference in their entrireties). Most strikingly, thechromosomes and a-tubulin (SEE FIG. 11J) were organized into ametaphase-like spindle (SEE FIG. 11K), which resembled a freshlyovulated, MII-arrested egg (SEE FIG. 11L-O). It should be noted thatonly eggs with a clear second polar body were selected for ZnPTtreatment; thus, the spindle configuration in ZnPT-treated activatedeggs, while metaphase-like, is not that of metaphase II. An analogousmetaphase-like spindle and arrest has been seen in parthenogeneticallyactivated rodent eggs that receive insufficient activating stimulus:such cells complete meiosis II but stall at a so-called third metaphase(MIII) instead of progressing to interphase (Kubiak. (1989) Dev Biol136, 537-45.; Zernicka-Goetz. (1991) Mol Reprod Dev 28, 169-76.; hereinincorporated by reference in their entireties).

In the reverse of the zinc-loading experiment, the intracellularavailability of zinc in the egg was decreased by continuous culture inthe presence of 10 μM TPEN. After 8 hrs of culture, most control eggsmaintained metaphase II arrest while those exposed to TPEN had apronucleus-like structure at significantly higher rates (SEE FIG. 12).These complementary experiments support the finding that a significantlyelevated zinc quota is associated with metaphase arrest and that thecoordinated exocytosis of zinc upon egg activation relieves this arrest.Furthermore, these results also distinguish the intracellular target ofthe calcium-selective chelator BAPTA AM (SEE FIG. 4) from that of TPEN.BAPTA is also known to have a substantial affinity for zinc (Haugland.,Handbook of fluorescent probes and research chemicals. 6th ed. MolecularProbes: Eugene, Oreg., 2001.; Grynkiewicz et al. (1985) J Biol Chem 260,3440-50.; Stork & Li. (2006) J Neurosci 26, 10430-7.; hereinincorporated by reference in their entireties). At the 10 μMconcentration used in experiments conducted during development ofembodiments of the present invention, BAPTA and TPEN had significantlydifferent effects on egg physiology. This is illustrated by the factthat long-term exposure to BAPTA did not parthenogenetically activateeggs (SEE FIG. 4) but TPEN did (SEE FIG. 12). Under these conditions,BAPTA is scavenging intracellular free calcium and thereby blockingcalcium oscillations. Taken together, the results support the findingthat calcium oscillations initiate and are required for the programmedloss of cellular zinc via the zinc sparks and that in turn drives cellcycle resumption.

EXAMPLE 6 Zn-Responsive Probe Synthesis

Experiments were conducted during development of embodiments of thepresent invention to synthesize a Zn-responsive probe comprising: aZn-chelating ligand containing five Zn-binding groups (three pyridylgroups and two tertiary amines), a BODIPY fluorophore that hasexcitation and emission wavelengths in the visible spectrum, an ethanolether based linker, and an amine group to enable attachment tocarboxylate-modified surfaces via amide coupling chemistries (SEE FIG.13). The starting material, Compound 1, is synthesized according toliterature procedures (Domaille et al. J. Am. Chem. Soc 2010, 132,1194-1195.; herein incorporated by reference in its entirety). Compound1 was combined with N-boc-ethanolamine and sodium hydride to yieldcompound 2. This was then heated in the presence of the Zn-binding group3 to furnish compound 4. Finally, the Boc group was removed fromcompound 4 using borontrifluoride diethyletherate to yieldamine-functionalized compound 5 (SEE FIG. 14). Experiments conductedduring development of the present invention demonstrated that the probesynthesized in Example 6 undergoes alteration in its detectable signal(BODIPY fluorescence) upon binding of Zn to the Zn-chelating ligand.

EXAMPLE 7 ZincBY-1 Probe Synthesis

This Example describes the synthesis of an exemplary probe calledZincBy-1, which is pictured in FIG. 15B. A schematic of this synthesisis shown in FIG. 15C. ZincBy-1 is readily synthesized in 70% yield fromstarting materials that have been previously reported (see, Domaille, etal. J. Am. Chem. Soc. 2010, 132, 1194-1195; and Hureau, et al. Inorg.Chem. 2008, 47, 9238-9247., both of which are herein incorporated byreference).

3-Chloro-5-methoxy-8-mesityl-BODIPY (32 mg, 0.085 mmol) andN¹-(pyridin-2-yl)-N²,N²-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (57mg, 0.17 mmol) were combined in 5 mL of dry CH3CN in an oven driedSchlenk flask under Na. The reaction was heated at 80° C. overnight.Following cooling and solvent removal, the product was purified bysilica gel chromatography (0-5% CH₃OH in CH₂Cl2) to yield the pureproduct as a dark pink residue (40 mg, 70% yield). ¹H NMR (CDCl₃, 500MHz): δ 8.51 (1H, m), 8.48 (2H, m), 7.62 (3H, dq, J=2.0, 7.5 Hz), 7.53(2H, d, J=8.0 Hz), 7.37 (1H, d, J=8.0 Hz), 7.15 (1H, m), 7.12 (2H, m),6.89 (2H, s), 6.34 (1H, d, J=4.5), 6.10 (1H, d, J=4.0 Hz), 5.87 (1H, d,J=4.5 Hz), 5.61 (1H, d, J=4.0 Hz), 5.22 (2H, s), 3.96 (3H, s), 3.89 (4H,s), 3.86 (2H, m), 2.92 (2H, m), 2.33 (3H, s), 2.08 (6H, s). ¹³C NMR(CDCl₃, 125 MHz): δ 164.51, 164.27, 160.03, 156.42, 148.03, 147,58,136.69, 136.39, 135.98, 131.91, 130.60, 129.37, 128,36, 126.37, 126.83,124.37, 121.59, 121.56, 121.39, 121.15, 110.10, 106.20, 52.44, 30.92,30.59, 29.96, 28.70, 21.70, 18.89, 13.14 ESI-MS MH⁺ C₃₉H₄₁BF₂N₇O⁺ calc.672.6, found 672.3.

EXAMPLE 8 ZincBY-1 Probe Characterization

This Example describes the characterization of the ZincBy-1 probe, whichis shown in FIG. 15B. ZincBY-1 has excellent in vitro spectroscopicproperties and can be used, for example, in live cells at nanomolarconcentrations (see below). ZincBY-1 was characterizedspectroscopically. Solutions of 5 μM of ZincBY-1 in 50 mM HEPES, 0.1 MKNO₃, pH 7.2 were analyzed for their absorbance and fluorescenceproperties (FIGS. 16 and 17). Absorbsbance properties of the apo- andzinc-bound forms of the probe reveal absorbance in the 500 to 600 nmrange, which is typical for BODIPY-based sensors (FIG. 16). Theextinction coefficient for the apo form is 12,000 cm⁻¹M⁻¹ and for theZn-bound form is 28,000 cm⁻¹M⁻¹. Fluorescence characterization (FIG. 17)with an excitation wavelength of 530 nm revealed minimal fluorescence inthe Apo form (quantum yield =0.02). Upon addition of 1 equiv ZnSO₄,fluorescence at 543 nm increases substantially, with a quantum yield of0.46. Taken together, these data reveal a ˜50-fold fluorescence turn-onfor Zn, which is comparable with the state-of-the-art commerciallyavailable probe FluoZin-3 (Gee, et al. J. Am. Chem. Soc. 2002, 124,776-778, which is herein incorporated by reference), which has a 80-foldturn on.

ZincBY-1 is selective for Zn²⁺ over a range of metal ions includingCa²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, and Cu⁺ (FIG. 18). Inthe absence of Zn, none of these metal ions induce an increase inZincBY-1 fluorescence. In the presence of Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Fe³⁺,Co²⁺, and Ni²⁺, ZincBY-1 is still capable of detecting Zn as addition ofZn induces a fluorescence increase. In the presence of Cu²⁻ and Cu⁺,however, ZincBY-1 does not respond to Zn addition, indicating that Cu²⁺and Cu⁺ bind ZincBY-1 more tightly than Zn²⁺ but quench ZincBY-1-basedfluorescence. In a cellular context, Zn is present in ˜10× excess of Cu,and thus, interference from Cu is not expected in cells.

Zn binding of ZincBY-1 occurs in a 1:1 fashion as is demonstrated by theJob plot in FIG. 19. In these experiments, the combined ZincBY-1 and Znconcentration is held constant, but the proportions of the twocomponents are varied. Maximum signal is observed at 0.5 mole fractionof both components, consistent with a 1:1 binding stoichiometry.ZincBY-1 binds zinc with nanomolar affinity as is shown in experimentswhere free Zn is buffered by EGTA (FIG. 20). The data is fitted to acurve that corresponds to K_(d)=4.4 nM, with a Hill slope of ˜2. In theabsence of zinc, ZincBY-1 displays constant fluorescence over a range ofpH values (pH 3.5 to 12.2, FIG. 21). The zinc response of ZincBY-1 isrobust over a pH range of pH 4 to 8, which matches well with thebiologically relevant pH range.

Following in vitro characterization of ZincBY-1, the probe was tested inlive MII mouse eggs. Live MII eggs were incubated in 50 nM ZincBY-1 and0.1 mg/mL Hoechst for 10 minutes and transferred to an imaging dish.FIG. 22 depicts a confocal z-stack of an MII egg, with greenrepresenting ZincBY-1 fluorescence (indicates the presence of Zn) andblue represents Hoechst fluorescence (labels DNA). In this context, 50nM ZincBY-1 is capable of labeling zinc-rich vesicles inside the cell.

All publications and patents mentioned in the present application and/orlisted below are herein incorporated by reference. Various modification,recombination, and variation of the described features and embodimentswill be apparent to those skilled in the art without departing from thescope and spirit of the invention. Although specific embodiments havebeen described, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes and embodiments that areobvious to those skilled in the relevant fields are intended to bewithin the scope of the following claims.

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We claim:
 1. A Zn-responsive probe which has the following chemicalstructure:

wherein each of said R_(a)s are individually selected from: H, CH₃, analkyl group, an O-alkyl group, an N-alkyl group, a linker group, anattachment group, and a linker group attached to an attachment group;wherein said R₁ is selected from: H, CH₃, an alkyl group, a linkergroup, an attachment group, and a linker group attached to an attachmentgroup; and wherein said R₂ is a Zn binding group.
 2. The Zn-responsiveprobe of claim 1, wherein said Zn binding group has the followingformula:

wherein A and B each seperately comprise at least one moiety selectedfrom an amine donor, an oxygen donor, and a sufur donor.
 3. TheZn-responsive probe of claim 1, wherein said Zn binding group has thefollowing formula:

wherein A and B each seperately comprise at least one moiety selectedfrom a pyridine, an amine, an imidazole, a sulfonamide, a carboxylate, aphenol, an ether, an alcohol, a thiol, a thioether, and a thiophene. 4.The Zn-responsive probe of claim 1, wherein said Zn binding group hasthe following formula:

or a derivative of said formula that is able to chelate zinc.
 5. TheZn-responsive probe of claim 1, wherein said chemical structure is asfollows:


6. The Zn-responsive probe of claim 1, wherein each of said R₃s are CH₃.7. The Zn-responsive probe of claim 1, wherein said R₁ is CH₃.
 8. TheZn-responsive probe of claim 1, wherein said R₁ comprises an attachmentgroup.
 9. A method of non-invasively detecting Zn release and/or uptakeby a cell or cells comprising: a) contacting a sample comprising a cellor cells with a composition comprising a Zn-responsive probe, whereinsaid Zn-responsive probe is present in said composition at aconcentration between 1×10⁻⁵ M and 1×10⁻⁹ M; b) detecting the signalemitted by the signaling moiety over time; and c) correlating signalemitted with Zn concentration.
 10. The method of claim 9, wherein thesample comprises one or more oocytes.
 11. The method of claim 9, whereinthe Zn-responsive probe is attached to a surface.
 12. The method ofclaim 9, wherein the concentration of said Zn-responsive probe isbetween 1×10⁻⁷ and 1×10⁻⁸ M.
 13. The method of claim 9, wherein saidcell comprises a metaphase-II-arrested oocyte, wherein said methodfurther comprises a step before step b) of contacting the sample withsperm, and wherein said detecting the signal comprises detecting asignificant increase in extracellular Zn concentration based on a changein the signal of the Zn-responsive probe.
 14. The method of claim 13,wherein said correlating said signal comprises correlating thesignificant increase in extracellular Zn concentration to fertilizationof the oocyte by the sperm.
 15. The method of claim 14, furthercomprising removing the oocyte from the Zn-responsive probe, andimplanting the fertilized oocyte into a subject.
 16. The method of claim9, wherein the Zn-responsive probe has the chemical structure shown inclaim
 1. 17. A method of evaluating the quality of an oocyte forfertilization comprising: a) contacting a surface with a samplecomprising the oocyte, wherein said surface displays a plurality ofZn-responsive probes; b) detecting the signal emitted by theZn-responsive probes over time; and c) correlating the signal emittedwith extracellular Zn concentration, wherein a decrease in extracellularZn is indicative of an oocyte that is ready for fertilization.
 18. Themethod of claim 17, where the oocyte is in prophase-I-arrest uponcontacting with the surface.
 19. The method of claim 17, wherein theZn-responsive probe is attached to a surface.
 20. The method of claim19, wherein the Zn-responsive probe has the chemical structure show inclaim 1.